B cells often constitute abundant cellular components in human tumors. Regulatory B cells that are functionally defi ned by their ability to produce IL10 downregulate infl ammation and control T-cell immunity. Here, we identifi ed a protumorigenic subset of B cells that constitutively expressed higher levels of programmed cell death-1 (PD-1) and constituted ∼ 10% of all B cells in advanced-stage hepatocellular carcinoma (HCC).

B cells consistently represent abundant cellular components in tumors; however, direct evidence supporting a role for B cells in the immunopathogenesis of human cancers is lacking, as is specific knowledge of their trafficking mechanisms. Here, we demonstrate that chemokine (C-X-C motif ) receptor 3-positive (CXCR3 1 ) B cells constitute approximately 45% of B-cell infiltrate in human hepatocellular carcinoma (HCC) and that their levels are positively correlated with early recurrence of HCC. These cells selectively accumulate at the invading edge of HCC and undergo further somatic hypermutation and immunoglobulin G-secreting plasma cell differentiation. Proinflammatory interleukin-17 1 cells are important for the induction of epithelial cell-derived CXCR3 ligands CXCL9, CXCL10, and CXCL11, which subsequently promote the sequential recruitment and further maturation of CXCR3 1 B cells. More importantly, we provide evidence that CXCR31 B cells, but not their CXCR3 -counterparts, may operate in immunoglobulin G-dependent pathways to induce M2b macrophage polarization in human HCC. Depletion of B cells significantly suppresses M2b polarization and the protumorigenic activity of tumor-associated macrophages and restores the production of antitumorigenic interleukin-12 by those cells in vivo. Conclusion: Selective recruitment of CXCR3 1 B cells bridges proinflammatory interleukin-17 response and protumorigenic macrophage polarization in the tumor milieu, and blocking CXCR3 1 B-cell migration or function may help defeat HCC. (HEPATOLOGY 2015;62:1779-1790 T he communications between cancer cells and immune elements might directly promote tumor development and progression and/or result in immunoediting of tumors that fosters immune privilege and/or chronic inflammation.1-3 The subset composition and function of myeloid cells and T cells have been extensively studied in human cancers.4 B cells are essential components of humoral immunity and act as both antigen-presenting cells and effector cells, 5 but the subset composition and the biological role of B cells are poorly defined in the human cancer microenvironment.As the clinical application of monoclonal antibodies increases, the antitumor effect of antigen-specific B cells receives increasing attention.6 Antibodies secreted byAbbreviations: B-cell-CM, 25% conditioned medium from tumor CXCR3 1 B

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Spatially Targeted Optical Micro Proteomics (STOMP) is a method to study region-specific protein complexity of a biological specimen. STOMP uses a confocal microscope to both visualize structures of interest and to tag the proteins within those structures by a photo-driven crosslinking reaction so that they can be affinity purified and identified by mass spectrometry 1 . STOMP has the potential to perform discovery proteomics on sub-cellular structures in a wide range of primary cells types and biopsy-scale tissue samples. However, two significant limitations have prevented the broad adoption of this technique by the scientific community. First, STOMP is performed across two software platforms written in different languages, which requires user operation at each field of view. Up to 48 hours of microscope time is necessary to tag sufficient protein (~1 μg) for mass spectrometry making STOMP prohibitively time and labor-consuming for many researchers. Second, the original STOMP protocol uses a custom photo-crosslinker that limits the accessibility of the technique for some user. To liberate the user, we developed a protocol that automates communication between Zeiss Zen Black imaging software and FIJI image processing software using a customizable code in SikuliX. To fully automate STOMP (autoSTOMP), this protocol includes a tool to make tile array, autofocus and capture images of fields of view across the sample; as well as a method to modify the file that guides photo-tagging so that subsets of the structures of interest can be targeted. To make this protocol broadly accessible, we implemented a commercially available biotin-benzophenone crosslinker as well as a procedure to block endogenous biotin and purify tagged proteins using magnetic streptavidin beads. Here we demonstrate that autoSTOMP can efficiently label, purify and identify proteins that belong to structures measuring 1-2 m in diameter using human foreskin fibroblasts or mouse bone marrow-derived dendritic cells infected with the protozoan parasite Toxoplasma gondii (Tg). The autoSTOMP platform can easily be adapted to address a range of research questions using Zeiss Zen Black microscopy systems and LC-MS protocols that are standard in many institutional research cores. INTRODUCTION:Localization is a critical regulatory component of protein function. Currently, there is a tremendous demand for biochemical and microscopy techniques to assess regional protein complexity and function in tissues, cells, and organelles. Spatially targeted optical micro proteomics (STOMP) was designed to identify the proteome of regional structures. 1 STOMP was first implemented to identify components of amyloid plaques in brain sections from a mouse model of prion disease and Alzheimer's disease patients. 1 In STOMP, structures of interest (SOI) are stained for fluorescence microscopy and identified by confocal imaging. Confocal images are used to generate a "MAP" file. Using a custom macro, the "MAP" file guides the 2-photon laser to revisit structures of interest and ...